U.S. patent application number 10/374828 was filed with the patent office on 2003-08-14 for charged bio-molecule/binding agent conjugate for biological capture.
Invention is credited to Sullivan, Brian M., Zsolnay, Denes L..
Application Number | 20030153024 10/374828 |
Document ID | / |
Family ID | 46282049 |
Filed Date | 2003-08-14 |
United States Patent
Application |
20030153024 |
Kind Code |
A1 |
Sullivan, Brian M. ; et
al. |
August 14, 2003 |
Charged bio-molecule/binding agent conjugate for biological
capture
Abstract
ELISA (and ELISA-like) procedures for detection of agents, such
as bioagents, proteins and nucleic acids, incorporate electrically
charged (3) recognition molecules (5) that bind to the specific
agent (7) being sought, deposit the charged recognition molecules
and bound suspect agent in a fluid, and incorporate an electric
field (E) to move and/or position those charged molecules to
specific locations within the solution during the immunoassay
procedure.
Inventors: |
Sullivan, Brian M.;
(Manhattan Beach, CA) ; Zsolnay, Denes L.;
(Rolling Hills Estates, CA) |
Correspondence
Address: |
PATENT COUNSEL, TRW INC.
S & E LAW DEPT.
ONE SPACE PARK, BLDG. E2/6051
REDONDO BEACH
CA
90278
US
|
Family ID: |
46282049 |
Appl. No.: |
10/374828 |
Filed: |
February 25, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10374828 |
Feb 25, 2003 |
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09837946 |
Apr 19, 2001 |
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6562209 |
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Current U.S.
Class: |
435/7.92 ;
205/777.5; 702/19 |
Current CPC
Class: |
G01N 35/00693 20130101;
G01N 27/48 20130101; G01N 2035/1025 20130101; G01N 35/0098
20130101; G01N 35/08 20130101; G01N 35/00594 20130101 |
Class at
Publication: |
435/7.92 ;
205/777.5; 702/19 |
International
Class: |
G01N 033/53; G01N
033/537; G01N 033/543; G06F 019/00; G01N 033/48; G01N 033/50 |
Claims
1. In a reporter system that detects the presence of a specific
biological agent by linking both an electrically charged
recognition molecule and a recognition molecule and enzyme complex
to the biological agent and introducing a substrate of the enzyme
to a liquid containing said biological agent linked electrically
charged recognition molecule and recognition molecule and enzyme
complex, wherein the enzyme cleaves the substrate to release a
reporter in said liquid, said reporter system including a sensor
for sensing the amount of said reporter in said liquid, the
improvement comprising: electric field generating means for
generating an electric field in the vicinity of said sensor to
attract said biological agent and linked electrically charged
recognition molecule and recognition molecule and enzyme complex to
the vicinity of said sensor.
2. The method of detecting a particular agent that comprises a
bioagent, protein, or nucleic acid, comprising the steps of: mixing
said agent in a solution containing an electrically charged
recognition molecule to said agent to form a 1.degree. recognition
molecule/agent complex; mixing a 2.degree. recognition molecule
enzyme complex together with said 1.degree. recognition
molecule/agent complex to form a 1.degree. agent/recognition
molecule/2.degree. recognition molecule enzyme complex; applying an
electric field to said 1.degree. agent/recognition
molecule/2.degree. recognition molecule enzyme complex for
positioning said 1.degree. agent/recognition molecule/2.degree.
recognition molecule enzyme complex at a reporter sensing location;
introducing a substrate of said enzyme to said 1.degree.
agent/recognition molecule/2.degree. recognition molecule enzyme
complex to cleave a reporter from said substrate at said reporter
sensing location; and sensing said reporter.
3. The method of detecting a particular agent that comprises a
bioagent, protein, or nucleic acid, as defined in claim 2, which
includes, prior to said step of mixing said agent in a solution
containing an electrically charged recognition molecule to said
agent to form a 1.degree. recognition molecule/agent complex, the
step of imparting an electrical charge to said recognition
molecule.
4. The method of detecting a particular agent that comprises a
bioagent, protein, or nucleic acid, as defined in claim 3, wherein
said step of imparting an electrical charge to said recognition
molecule, includes the step of molecularly splicing a polyglutamate
to said recognition molecule.
5. Apparatus for conducting an electrochemical enzyme linked
immunosorbent assay ("ELISA") for a bioagent, protein or nucleic
acid comprising: a plurality of vessels, said plurality of vessels
including: a first vessel for holding electrically charged
recognition molecules in liquid; a second vessel for holding a wash
solution; a third vessel for holding a 2.degree. recognition
molecule linked enzyme; a fourth vessel for holding a substrate
reporter; and sample holding means for holding a sample solution
containing said bioagent, protein or nucleic acid; an examination
cell; an electronic controller, said electronic controller
including a program, a start switch and a display; a sensor for
electrically detecting the level of reporter present in said
examination cell at any moment of time and supplying said detected
level of reporter present at any moment in time to said electronic
controller; an electric field generator controlled by said
electronic controller for producing an electric field inside said
examination cell when required by said program; said electronic
controller for motivating passage of the respective contents of
each of said vessels into said examination cell when required by
said program and for motivating removal of the contents of said
examination cell in whole and/or in part when required by said
program; said program further defining an ELISA, wherein motivation
of each of said vessels is motivated in a sequence to pass contents
of the respective vessel into said examination cell to perform an
ELISA; said program defining said ELISA including means for
motivating the contents of said fourth vessel into said examination
cell and initiating assembly of the detected level of reporter
present at each of a plurality of time intervals from said sensor;
whereby insertion of the contents of said fourth vessel into said
examination cell when said bioagent is present in said examination
cell produces an electrochemical redox recycling reaction inside
said examination cell to produce levels of reporter that increases
with time; wherein said ELISA includes means for washing the fluid
in said examination cell; said means for washing including: first
means for motivating said electric field generator to produce an
electric field in said examination cell responsive to a command
from said electronic controller, wherein said electrically charged
recognition molecules in said examination cell are drawn to one
side of said examination cell leaving a portion of said examination
cell free of recognition molecules; second means for aspirating
fluid from said region of said examination cell vacated by said
recognition molecules while said electric field is present
responsive to a command from said electronic controller, and third
means for motivating wash fluid from said second vessel into said
examination cell responsive to a command by said electronic
controller following aspiration of fluid by said second means; said
program further including an analysis program for analyzing the
detected level of reporter at each of said plurality of time
intervals and determining the concentration of bioagent present in
said sample when said bioagent is present in said sample and
displaying said concentration on said display.
6. The apparatus for conducting an electrochemical ELISA for a
bioagent, protein or nucleic acid as defined in claim 5, wherein
said means for motivating the contents of said fourth vessel into
said examination cell and initiating assembly of the detected level
of reporter present at each of a plurality of time intervals from
said sensor further includes: means for motivating said electric
field generator to produce an electric field in said examination
cell that extends through said sensor, wherein said electrically
charged recognition molecules in said examination tube are drawn to
said sensor.
7. Apparatus for conducting an electrochemical enzyme linked
immunosorbent assay ("ELISA") for a bioagent, protein or nucleic
acid comprising: a plurality of vessels, said plurality of vessels
including: a first vessel for holding 1.degree. antibodies in
liquid; a second vessel for holding a wash solution; a third vessel
for holding a 2.degree. antibody linked enzyme; a fourth vessel for
holding a substrate reporter; and sample holding means for holding
a sample solution containing said bioagent; an examination cell; a
plurality of electric pumps, each pump being associated with a
respective one of said first through fourth vessels and said sample
holding means for conveying contents from the respective vessel
into said examination cell when said pump is energized; an
electronic controller, said electronic controller including a
program, a start switch and a display; said electronic controller
being coupled to said plurality of pumps for controlling the
energization of said pumps in accordance with said controller
program; said electronic controller further including a look-up
table, said look-up table containing a plurality of numbers
defining slopes and a plurality of bioagent concentrations with
each of said plurality of bioagent concentrations being associated
with a respective one of said plurality of numbers, wherein for
each slope represented in said look-up table, a concentration of
said bioagent may be determined; an aspirating pump, said
aspirating pump for pumping contents from said examination cell
when energized by said electronic controller; an electric field
generator controlled by said electronic controller for producing an
electric field inside said examination cell when required by said
program; a sensor for electrically detecting the level of reporter
present in said examination cell at any moment of time and
supplying said detected level of reporter present at any moment in
time to said electronic controller, said sensor for producing an
electrical current that is proportional to the quantity of reporter
present in said examination cell, whereby the electrical current
level increases as the amount of reporter developed with time in
said examination cell increases; said sensor further comprising a
pair of spaced comb-like shaped electrodes interdigitally arranged,
said spaced electrodes being located inside said examination cell
to provide a current carrying path in a gap between said electrodes
through at least a portion of the contents of said examination
cell, and a pair of electrical conductors for respectively
connecting each of said spaced electrodes to a source of potential
external to said examination cell; said electronic controller for
motivating passage of the respective contents of each of said
vessels into said examination cell when required by said program
and for motivating removal of the contents of said examination cell
in whole and/or in part when required by said program; said program
further defining an ELISA, wherein motivation of each of said
vessels is motivated in a sequence to pass contents of the
respective vessel into said examination cell to perform an ELISA;
said ELISA including means for washing the fluid in said
examination cell, said means for washing further comprising: first
means for motivating said electric field generator to produce an
electric field in said examination cell responsive to a command
from said electronic controller, wherein electrically charged
recognition molecules in said examination cell are drawn toward a
side of said examination cell leaving a portion of said examination
cell vacant of recognition molecules; second means for aspirating
fluid from said bead vacated region of said examination cell while
said electric field is present responsive to a command from said
electronic controller, and third means for motivating wash fluid
from said second vessel into said examination cell responsive to a
command by said electronic controller following aspiration of fluid
by said second means; said program defining said ELISA including
means for motivating the contents of said fourth vessel into said
examination cell, motivating said electric field generator to
produce an electric field in said examination cell that extends
through said sensor to draw electrically charged recognition
molecules in said examination tube to said sensor, and initiating
assembly of the detected level of reporter present at each of a
plurality of time intervals from said sensor; whereby insertion of
the contents of said fourth vessel into said examination cell when
said bioagent is present in said examination cell produces an
electrochemical reaction inside said examination cell to produce
levels of reporter that increases with time; said program further
including an analysis program for analyzing the detected level of
reporter at each of said plurality of time intervals and
determining the concentration of bioagent present in said sample
when said bioagent is present in said sample and displaying said
concentration on said display; said analysis program including: a
regression analysis program for performing a least-square linear
regression analysis on said detected level of reporter taken at
each of said plurality of time intervals to determine a number,
said number defining a slope; a look up program for looking up said
number determined by said regression analysis program in said
look-up table and locating the corresponding concentration of said
bioagent represented thereby.
8. The method of fabricating an electrically charged recognition
molecule comprising the steps of: molecularly splicing a string of
DNA that codes for a polyglutamate into a proteinaceous recognition
molecule of known DNA sequence to produce a combined DNA sequence,
said recognition molecule being composed of amino acids; and
inserting said combined DNA sequence into a producer cell, wherein
said producer cell produces electrically charged recognition
molecules.
Description
REFERENCE TO PRIOR APPLICATIONS
[0001] Reference is made to U.S. application, Ser. No. 09/837,946,
filed Apr. 19, 2001, entitled "Automated Computer Controlled
Reporter Device for Conducting Immunoassay and Molecular Biology
Procedures," of which the present invention is a continuation in
part. Applicant claims the benefit of 35 U.S.C. .sctn.120 based on
the foregoing application.
FIELD OF THE INVENTION
[0002] This invention relates to automated reporter systems that
control the movement and position of recognition molecules in the
performance of immunoassays of bioagents and other molecular
biology test procedures used to detect bioagents and to make
diagnoses, and, more particularly, to a method and apparatus for
controlling the movement and positioning of recognition molecules,
such as antibodies, suspended in fluid, used in those reporter
systems.
BACKGROUND
[0003] Controlling the motion of a recognition molecule in process
apparatus for conducting immunoassay and other molecular biology
test procedures, referred to herein as automated reporter systems,
and otherwise is not new. For one, that controlled motion is
accomplished in a prior apparatus, created by the present
inventors, that is described in U.S. application, Ser. No.
09/837,946, filed Apr. 19, 2001, entitled "Automated Computer
Controlled Reporter Device for Conducting Immunoassay and Molecular
Biology Procedures." An embodiment of the automated apparatus
disclosed in the cited application detects specific bioagents using
an enzyme linked immunoassay, referred to as an "ELISA" process.
That immunoassay requires a recognition molecule, such as an
appropriate antibody, that attaches to (i.e. adsorbs) a suspect
bioagent molecule. To control the position of the recognition
molecule in a liquid solution during that ELISA process, the
molecule is coated on a magnetic bead, which serves as a carrier
for the recognition molecule in the solution. A permanent magnet in
the apparatus produces a magnetic field that is used to move and
position the coated magnetic bead within the vessel that holds the
liquid solution, and, hence, the recognition molecule, to locations
required during the performance of the immunoassay procedure.
[0004] The ELISA process constitutes an identification process that
uses molecular interactions to uniquely identify target substances.
A basic definition of ELISA is a quantitative in vitro test for an
antibody or antigen (e.g., a bioagent) in which the test material
is adsorbed on a surface and exposed to a complex of an enzyme
linked to an antibody specific for the substance being tested for
with a positive result indicated by a treatment yielding a color in
proportion to the amount of antigen or antibody in the test
material. Color and color intensity serves as the reporter or
indicia of the antigen or antibody. The basic ELISA procedure is
described more specifically, for one, in a book entitled Methods in
Molecular Biology Vol. 42, John R. Crowther, Humana Press,
1995.
[0005] The "antibody specific for the substance being tested for"
in the foregoing definition constitutes a recognition molecule, a
molecule that is capable of binding to either reactant or product
molecules in a structure-restricted manner. That is, the
recognition molecule binds to a specific three-dimensional
structure of a molecule or to a two-dimensional surface that is
electrically charged and/or hydrophobic in a specific surface
pattern. It may also be recognized that the same essential
approach, referred to herein as ELISA-like, can also be used using
other recognition molecules, such as aptamers, DNA (e.g.,
deoxyribonnucleic acid), RNA and molecular imprint polymers.
[0006] More recently, the foregoing definition of ELISA has been
expanded beyond the colormetric approach to include a voltametric
or amperiometric approach to detection and assay. In the latter,
the rate of change of voltage or current conductivity is
proportional to the amount of antigen or antibody contained in the
test sample. Such an approach is found in published PCT application
PCT/US98/16714, filed Aug. 12,1998 (International Publication No.
WO 99/07870), "Electrochemical Reporter System for Detecting
Analytical Immunoassay and Molecular Biology Procedures" (hereafter
the "16714 PCT application"). That published application describes
both a colormetric and an electrochemical reporter system for
detecting and quantifying enzymes and other bioagents in analytical
and clinical applications. The electrochemical reporter system of
the 16714 PCT application employs a sensor for detecting
voltametric and/or amperiometric signals that are produced in
proportion to the concentration of organic (or inorganic) reporter
molecules by redox (e.g., reduction-oxidation) recycling at that
sensor.
[0007] In brief, in the run of the ELISA process, the suspect
bioagent is initially placed in a water-based buffer, such as a
phosphate buffered saline solution, to form a sample solution. That
sample solution is mixed with a quantity of particles, such as
beads, coated with an antibody to the suspect bioagent (e.g., the
recognition molecule, also sometimes referred to as a receptor
molecule). The particular antibody used to coat the beads is one
that is known to bind to the bioagent of interest (e.g., the target
molecule) and is a primary antibody or "1.degree. Ab." Binding is
the chemical "stickiness" that is selective to specific
bioagents.
[0008] Any bioagent that is present in the sample solution binds
with a non-covalent bond to a respective recognition molecule
(e.g., antibody) and thereby becomes attached through the
recognition molecule to a respective one of the magnetic beads in
the sample solution. If the sample solution does not contain a
bioagent or if the bioagent that is present in the solution is not
one that binds to the selected antibody, then the recognition
molecule on the bead remains unbound or free. Continued processing
of the ELISA process in that case reports nothing. However, if the
suspect bioagent is present in the sample solution, the bioagent
binds to the recognition molecule coated on the beads. What then
results is a quantity of bioagent molecules indirectly bound
(through the recognition molecule), respectively, to a like
quantity of coated beads. The mixture is optionally washed, as
example, in a phosphate-buffered saline, and a second antibody,
more specifically, an antibody and enzyme linked combination, is
then added to the mixture.
[0009] The second antibody is also one that is known to bind to the
suspect bioagent, and constitutes another recognition molecule,
which may, but need not, be identical to the first antibody. The
second antibody may either be one that is monoclonal, e.g., one
that binds to only one specific molecule, or polyclonal, e.g., a
mixture of different antibodies each of which shares the
characteristic of bonding to the target bioagent. The enzyme is
covalently bound to the second antibody and forms a complex that is
referred to as a secondary antibody-enzyme conjugate or "2.degree.
Ab-enz." As known by those skilled in the art, an enzyme is a
"molecular scissors," a protein that catalyzes a biological
reaction, a reaction that does not occur appreciably in the absence
of the enzyme. The enzyme is selected to allow the subsequent
production of an electrochemically active reporter.
[0010] The2.degree. Ab-enz binds to the exposed surface of the
immobilized bioagent to form an "antibody sandwich" with the
bioagent forming the middle layer of that sandwich. The antibody
sandwich coated beads are washed again to remove any excess
2.degree. Ab-enz in the solution that remains unbound.
[0011] The magnetic beads and the attached antibody sandwiches, the
1.degree. Ab/bioagent/2.degree. Ab-enz complex, in the solution are
placed over the exposed surface of the redox recycling sensor. The
substrate of the foregoing enzyme is added to the solution and the
substrate is cleaved by the enzyme to produce an electrochemically
active reporter. The substrate of the enzyme, referred to as
PAP-Docket GP, is any substance that reacts with an enzyme to
modify the substrate. The effect of the enzyme is to separate or
cut the PAP, a para-amino phenol, the electrochemically active
reporter, from the GP, an electrochemically inactive substance.
[0012] The foregoing chemical reaction is concentrated at the
surface of the sensor. The rate of production of the foregoing
reporter (e.g., the PAP) is proportional to the initial
concentration of bioagent. The reporter reacts at the surface of
the sensor, producing an electrical current through the sensor that
varies with time and is proportional to the concentration of the
bioagent, referred to as redox recycling. The occurrence of the
electric current constitutes a positive indication of the presence
of the suspect bioagent in the sample. Analysis of the electric
currents produced over an interval of time and comparison of the
values of that electric current with existing laboratory standards
of known bioagents allows quantification of the concentration of
bioagent present in the initial sample.
[0013] The automated apparatus of the '946 application, hereafter
sometimes referred to as the automated ELISA system, provides a
user friendly stand-alone portable system that automatically
performs the ELISA process. The automated ELISA system contains a
number of solutions in respective reservoirs and pumps that are
controlled by a programmed computer. The electronic controller,
such as a programmed microcontroller, controls a series of electric
pumps to automatically sequence the pumping of the individual
solutions required by the ELISA procedure into and out of a cell
(or cells) as required by the ELISA program. That automated ELISA
system uses coated beads of magnetic material and a magnetic
positioning device to manipulate and position the coated magnetic
beads under control of the computer, such as during the washing
steps of the ELISA process, and in positioning the beads at the
sensor during the redox recycling that yields the PAP. The
controller commands the steps necessary to produce the reporter,
controls the positioning of the carrier of the reporter adjacent
the reporter sensor, analyzes the data obtained from the reporter
sensor and displays the concentration of the bioagent determined
from the analysis of the foregoing data. Once started, the
apparatus, governed by the program, conducts the test automatically
without the necessity for human intervention.
[0014] In a first step of the assay procedure, the sample solution,
containing the sample that is to be tested for the presence of a
specific bioagent, is placed in a container (or equivalent vessel)
that holds the 1.degree. antibody coated magnetic beads. If the
sample is of the specific bioagent, then the respective parts of
the sample links, sticks to, the antibody coating of a respective
bead.
[0015] For example, the sample solution that is to be tested for
the presence of a specific bioagent and the coated magnetic beads
are pumped from respective reservoirs into a container by
electrical pumps and mixed to ensure that the respective parts,
that is, molecules, of the sample contacts the coating of a
respective bead. In practice, the volume of the sample and bead
solution is small and the container, which may be a length of
pipette tubing, is also small. A known practical way to mix the
ingredients of the solution is to create turbulence by repeatedly
pumping the foregoing solution out of the container and then
pumping that solution back into the container or to recirculate
that solution.
[0016] Some of the sample may be unattached to a bead and that
excess needs to be removed from the solution by washing. To wash
the mixture, the magnetic beads (and attached molecules) are pulled
by a magnetic field controlled by the controller to one side of the
container, vacating a portion of the solution. An aspirating pipe
is immersed in that vacated portion of the solution. The controller
causes pumps to remove the dirty solution through the aspirating
pipe and to replace the dirty solution with clean solution, thereby
washing the magnetic beads.
[0017] The magnetic beads are preferably micron sized.
Collectively, the quantity of micron sized magnetic beads in the
solution appear as sludge; like mud in appearance and consistency,
and is abrasive. Such sludge like collection of beads is known to
be exceptionally hard on the valves and pumps of the automated
apparatus, prematurely reducing the operational life of those
valves and pumps. From the standpoint of improving the operational
life of the valves and pumps, the elimination of the abrasive
magnetic beads is clearly desirable. However, without those
magnetic beads, the automated apparatus could not function. Until
the present invention, no way of eliminating the magnetic beads was
known. As an advantage, the present invention provides an automated
apparatus that eliminates the magnetic beads yet controls the
movement of the recognition molecules. The operational life of the
pumps and valves in the automated apparatus is enhanced.
[0018] Accordingly, a principal object of the present invention is
to increase the operational life of the pumps and valves of the
automated ELISA apparatus.
[0019] An additional object of the invention is to eliminate the
magnetic beads from the automated ELISA apparatus while retaining
the ability of the apparatus to control the movement and
positioning of the recognition molecules during operation of the
apparatus.
[0020] A further object of the invention is to permit an applied
electric field to move and position the recognition molecules.
[0021] And a still further object of the invention is to compound
or otherwise create or build recognition molecules that are
electrically charged.
SUMMARY OF THE INVENTION
[0022] In accordance with the foregoing objects, ELISA (and
ELISA-like) procedures employs electrically charged recognition
molecules which, by design, bind to a specific bioagent. Those
bioagent bound charged recognition molecules are positioned within
a confined solution by applying an electrostatic field (e.g.,
electric field) to the solution to attract those charged molecules
to a desired location within the vessel confining that solution. In
the reporting stage of the ELISA and ELISA-like procedure the
bioagent bound charged recognition molecules are further bound to a
recognition molecule linked to an enzyme to form the recognition
molecule/bioagent/recognition molecule enzyme complex. Due to the
electric charge on the first recognition molecule in that complex,
the complex may be moved by the electric field to a position within
the solution adjacent a reporter sensor. When the substrate of the
enzyme, the PAP-GP molecules, is introduced into the solution, the
enzyme cuts or releases the PAP molecules (e.g., the reporter
molecules) from the PAP-GP molecule at a location adjacent the
sensor. The sensor detects the reporter molecules, and, indirectly,
detects the presence and concentration of the suspect bioagent
molecules that are present in the solution through changes in
resistivity of the solution produced by those reporter
molecules.
[0023] In accordance with a specific aspect to the invention,
recognition molecules are given a significant electrical charge by
attaching a polyglutamate to each recognition molecule using
standard molecular biological techniques, and those charged
molecules are deposited in a buffer solution. The inherent
electrical charge of the polyglutamates becomes an electrical
charge associated with each recognition molecule and polyglutamate
linked combination molecule.
[0024] The incorporation of the electrically charged recognition
molecules within the automated apparatus provides additional
benefit. The electrically charged recognition molecules are smaller
and weigh less than the corresponding micron sized recognition
molecule coated magnetic beads used in the prior automated ELISA
apparatus. By eliminating the greater bulk of the magnetic beads,
the pumps, valves and containers can be made smaller in size,
permitting greater miniaturization of the automated apparatus.
Thus, not only is the operational life of the pumps and valves
enhanced by eliminating the inherently abrasive magnetic beads, but
the automated apparatus is smaller and lighter in weight than the
earlier apparatus. That miniaturization enhances the overall
utility of the automated apparatus.
[0025] The foregoing and additional objects and advantages of the
invention, together with the structure characteristic thereof,
which were only briefly summarized in the foregoing passages, will
become more apparent to those skilled in the art upon reading the
detailed description of a preferred embodiment of the invention,
which follows in this specification, taken together with the
illustrations thereof presented in the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] In the drawings:
[0027] FIG. 1 is a pictorial illustration of the principal steps
carried out by the principal embodiment of the invention;
[0028] FIG. 2 is a pictorial illustration of the electrostatic
field structure used in the process of FIGS. 1 and FIGS. 3-5;
[0029] FIG. 3 is a block diagram of an embodiment of the
invention;
[0030] FIG. 4 is a block diagram of a second embodiment of the
invention; and
[0031] FIG. 5 is a partial schematic diagram of a third embodiment
of the invention that improves upon the embodiment of FIG. 4.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0032] FIG. 1, to which reference is made, pictorially illustrates
the various steps of an automated ELISA process that incorporates
the improvements of the present invention. The description of this
figure introduces the principles involved in this new process and
apparatus. With an understanding of the general description
accompanying this FIG. 1, the structure of the embodiments of FIGS.
3 through 5, later described, should be more readily understood and
the operation more easily followed. In FIG. 1 STEPS 1 through 6 of
the process are arranged in columns, left to right.
[0033] In preparation for the testing, a sample solution is
produced by placing the suspect material, bioagent 1, represented
in the figure by the pentagonal shaped symbol, in a water-based
buffer, such as a phosphate buffered saline solution. The foregoing
sample solution is to be tested for the presence of a specific
bioagent or may be a solution that is simply suspected of
containing a certain bioagent. If desired, the suspect material may
be preliminarily treated, such as by exposing the material to
ultrasonic energy, which breaks the material into multiple small
clumps or even granules, which ensures maximum surface area
exposure of the sample when placed in solution.
[0034] As further preparation, a supply of electrically charged
recognition molecules is produced using known molecular biological
techniques and then placed in a solution. Recognition molecules 5
are known to bind to the bioagents of interest (or of concern) on
contact with those bioagent molecules. That is, the recognition
molecule exhibits a chemical "stickiness" that is selective to
specific bioagents. The recognition molecule may be a single-chain
variable fragment ("sFv") molecule or monoclonal antibody of known
DNA (e.g., nucleic acid) sequence and is functionally equivalent to
a primary antibody or "1.degree. Ab." That known DNA sequence is
one to produce a molecule that binds or attaches to a specific
target molecule, such as bioagent 1. Such monoclonal antibodies are
typically grown from the cells of a mouse using known molecular
immunology techniques.
[0035] The extent of electrical charge inherently associated with a
given protein varies and is slight. The website of the Nanogen
company, www.nanogen.com, notes that biological molecules possess
natural positive or negative charges and may be attracted by an
area of opposite charge. That company purports to employ such
natural charges in DNA to permit the DNA to be captured on the
surface of a semiconductor chip containing appropriately placed
electrical charges, and then be operated upon. However, the slight
charge of proteins is insufficient for the immunoassay process
described herein, and is not regarded herein as being electrically
charged as that term is used herein. Hence, the recognition
molecule is modified to increase the electrical charge associated
with the molecule, either positively or negatively in charge,
beyond the inherent charge. That increase in electrical charge is
accomplished by splicing the DNA that codes for a polyglutamate
onto the monoclonal antibody, such as described herein in the final
paragraphs of this specification.
[0036] Glutamates are known to inherently possess a more robust
electrical charge. If more than one glutamate is attached in series
(e.g., a polyglutamate) the net electrical charge increases roughly
proportionately. Thus, the DNA code of the ultimate biological
"product," the electrically charged recognition molecule, contains
two DNA segments, each segment individually characterizing
something different. In this circumstance, the segments
respectively characterize the monoclonal antibody that serves as
the recognition molecule and the polyglutamate. In simplified
terms, the foregoing splicing process produces a recognition
molecule 5 that may be referred to as containing a "tail,"
represented by the lantern shaped symbol 3 in the figure, of
electric charge. The level of that electric charge is a function of
the number of glutamate molecules used to form that tail.
[0037] The electrically charged recognition molecule is deposited
in a fluid, such as deionized water that is buffered with some
salts, as example, sodium phosphate to control the pH of the
solution and sodium chloride to control ionic strength. Due to the
electrically charged character of the tails, the antibody 5 and
tail 3 combination can be manipulated in the solution by an
externally applied electric field.
[0038] Continuing with FIG. 1, in the first step of the test, STEP
1, sample solution 1 containing the bioagent is placed into a
container containing a quantity of the electrically charged
recognition molecules 5, here antibodies, to the suspect bioagent,
and mixed. The electrically charged tails 3 to those antibodies, in
this example, contain a negative charge (relative to earth or
ground).
[0039] Any bioagent molecule present in the solution binds to a
respective antibody 5 and thereby becomes attached to a respective
one of the electrically charged antibodies in the solution to form
a 1.degree. Ab/bioagent conjugate. The foregoing bond between the
antibody and bioagent is recognized as a non-covalent bond. As
becomes apparent, if no bioagent is present in the sample solution
or if the bioagent that is present in the solution is not one that
binds to the selected antibody 5, then antibody 5 remains unbound.
The results obtained from further processing as described herein
will then show nothing. For purposes of the description of
operation, the sample solution being tested is presumed to carry
bioagent 1.
[0040] Accordingly, bioagent 1 bonds to antibody 5, as represented
in STEP 3 by the seating of the pentagonal shaped bioagent symbol
inside the mating cavity of the antibody symbol. On conclusion of
STEP 1, the solution contains a quantity of bioagent molecules 1
bound respectively to a like quantity of electrically charged
antibodies 5.
[0041] In STEP 2 the mixture in solution of STEP 1 is optionally
washed before proceeding with step 3. A suitable wash solution, as
example is phosphate-buffered saline. Variants of the foregoing
wash solution may include additional components, such as bovine
serum albumin or detergents. To wash the mixture in solution, an
electric field, E, is first established and applied to the mixture.
That electric field is either produced external of the vessel or
other containment region holding the solution and is directed from
a field electrode through a side of the vessel. The foregoing
requires that the vessel or other containment region be constructed
of dielectric material through which static electric fields may
effectively extend, such as glass, ceramic or nylon.
[0042] Alternatively, the electric field may be produced inside the
vessel, in which case, the field electrode or electrodes are
located inside the vessel or confinement region, immersed in the
solution.
[0043] Assuming the former electrode structure, the electric field,
E, produced by electrode 11 effectively penetrates the walls of the
vessel and attracts the negatively charged antibodies to the side
closest to the positive electrode, leaving the solution at the
other side of the vessel relatively free of the charged antibodies.
The side of the solution in the vessel vacated by the antibodies is
then aspirated to remove the solution, suitably using an aspirating
tube, without removing the charged antibodies. The removed solution
is then replaced with clean solution. The renewed solution is then
agitated to re-suspend the antibodies 5 in the wash solution. The
foregoing agitation is preferably accomplished by pumping fluid
into and out of the reaction area of the vessel.
[0044] The structure for the foregoing operation is pictorially
illustrated in FIG. 2 to which reference is made. Container 2
contains a fluid containing the bioagent and recognition molecule
combination, aspirating tube 4, a sensor 13. The container also
supports a pair of field electrodes 11 and 11B, located on
diametrically opposite outer walls of the container. Sensor 13,
comprising interdigitated electrodes, as example, is positioned
along a wall of the vessel. Electrical leads 4 connected to the
respective electrodes of the sensor extend through a wall for
connection to the current monitoring apparatus, not illustrated,
and to a voltage extended from that monitoring apparatus.
[0045] Field electrodes 11 and 11B are electrically connected to
the opposite polarity terminals of a field voltage supply 32. The
field voltage supply is ungrounded and electrically isolated from
the voltage source, not illustrated, that is associated with the
current monitoring apparatus, referred to previously.
[0046] Field voltage supply 32 is switchable between an "on" and
"off" condition. When an appropriate switching signal or command is
applied at input C of the field voltage supply, the supply
generates the requisite voltage which appears between field
electrodes 11 and 11B, with the polarity of electrode 11 being
positive relative to electrode 11B. That voltage establishes the
electric field E that extends from electrode 11B through the walls
of container 2 and the solution, the opposite side of the container
wall to electrode 11. The field is essentially a DC electrostatic
field. The path between the electrodes is of very high resistance
and virtually no current is drawn from field voltage supply unit
32. Typically, the voltage produced by the field voltage supply is
under one volt, to avoid electrolysis of the water as could produce
hydrogen and oxygen gases, a rather explosive combination. The
negatively charged matter in the solution, charged antibody 5-3, is
attracted to one side by the electric field, the left side in the
figure, and vacates the opposite side, the right, of the container.
The liquid of the solution may then be aspirated by a pump via
aspirating tube 4, leaving the charged antibody alone; and the
withdrawn liquid may be replenished by pumping clean liquid through
that tube.
[0047] When the switching signal is removed or changed from the
input C of the field voltage supply, the electric field voltage
supply switches off and the electric field collapses, releasing the
attractive force on the charged antibody combination and allowing
that matter to circulate freely in the solution. As later herein
described, in the automated system the field voltage supply 32 and
the current monitoring apparatus associated with sensor 13 are
controlled by a computer. Sensor 13 does not serve any function
during the wash procedure, but performs a function in the later
stages of the test process, later herein described.
[0048] Returning to FIG. 1, following the foregoing wash procedure
of STEP 2, the electric field is again applied to again trap the
charged antibodies and the wash process is repeated. The foregoing
washing procedure is repeated as many times as experience shows is
necessary to adequately clean the solution. As those skilled in the
art appreciate, other washing protocols may be substituted for that
described using the electric field without departing from the
invention, but the electric field approach is believed most
convenient.
[0049] In the next step, STEP 3, a second recognition molecule,
more specifically, an antibody 7 and enzyme 9 linked combination-is
added to the mixture in solution. The second antibody 7 is also one
that is known to bind to bioagent 1 of interest. That antibody need
not be the same structure as the first antibody, and may be either
be one that is monoclonal, e.g., one that binds to only one
specific molecule, or polyclonal, e.g., a mixture of different
antibodies each of which shares the characteristic of bonding to
the target bioagent. The enzyme 9, illustrated by the scissors
symbol, is covalently bound to the second antibody 7 and forms a
complex that is referred to as a secondary antibody-enzyme
conjugate or "2.degree. Abenz."As is known, an enzyme is a
"molecular scissors," a protein that catalyzes a biological
reaction, a reaction that does not occur appreciably in the absence
of the enzyme. Enzyme 9 is selected so as to allow the subsequent
production of an electrochemically active reporter, described
hereafter in succeeding steps of the process.
[0050] The 2.degree. Ab-enz conjugate binds to the exposed surface
of the immobilized bioagent to form a charged 1.degree.
Ab/bioagent/2.degree. Ab-enz complex, an "antibody sandwich." The
bioagent forms the middle layer of that sandwich, such as is
illustrated in the pictorial of STEP 5 to which brief reference is
made. Returning to completion of STEP 3, the next step, STEP 4, is
to wash the charged antibody sandwich in a manner similar to, but
not necessarily identical to that described in STEP 2. At this
juncture in the test procedure, the purpose of the wash is
principally to wash away any excess 2.degree. Ab-enz that is not
bound to a bioagent. If one employs a separate reaction chamber (as
appears in one of the embodiments later described) and
electrochemical cell instead of a single cell, following the
foregoing washing, the charged 1.degree. Ab/bioagent/2.degree.
Ab-enz complexes are transferred from such reaction chamber to the
electrochemical cell prior to undertaking STEP 5.
[0051] In STEP 5, electrode 11 again produces electric field, E,
such as is represented by the arrow in the illustration. The
electric field is oriented to attract the electrically charged
antibody sandwich, the 1.degree. Ab/bioagent/2.degree. Ab-enz
complex, in the solution to the exposed surface of the sensor 13.
That sensor may be the redox recycling sensor consisting of
interdigitated electrodes, such as described in the published 16714
PCT application, earlier cited. Referring again to FIG. 2, at this
stage, field voltage supply 32 is set "on" to generate the electric
field. It is seen that the electric field E produced between
electrodes 11 and 11b extends through the interdigital finger
electrodes of sensor 13 and from those finger electrodes to
electrode 11.
[0052] Next, in STEP 6 the substrate 10 of the enzyme is added to
the solution and the substrate is cleaved by enzyme 9 to produce an
electrochemically active reporter. The substrate of the enzyme is
any substance that reacts with an enzyme to modify the substrate.
This is illustrated in STEP 6 in which a preferred embodiment of
substrate 10 is denominated PAP-GP. The effect of the enzyme is to
cut the PAP, a para-amino phenol, the electrochemically active
reporter, from the GP 12, an electrochemically inactive
substance.
[0053] The electric field produced by electrode 11 concentrates the
foregoing chemical reaction at the surface of the sensor. The rate
of production of the foregoing reporter (PAP) is proportional to
the initial concentration of bioagent. The reporter reacts at the
surface of electrochemical sensor electrodes 13, producing an
electrical current through the sensor electrodes that varies with
time and is proportional to the concentration of the bioagent.
[0054] An analysis of the electric currents produced in the
foregoing manner over an interval of time and comparison of the
values of that current with existing laboratory standards of known
bioagents allows quantification of the concentration of bioagent
present in the initial sample. More specifically, a least-square
linear regression analysis of the data generates the slope of the
current, representing the rate of change of current over time. That
slope is then compared with corresponding slopes that were
previously obtained in measurements of standard concentrations of
the bioagent. By selecting the closest match between the measured
and reference slopes the amount of bioagent present in the initial
sample is determined.
[0055] As those skilled in the art appreciate, the foregoing method
(and apparatus) of FIGS. 1 and 2 modifies the prior automated ELISA
procedure of the cited co-pending application Ser. No. 09/837,946,
filed Apr. 19, 2001, for one, by incorporating into the process
electrically charged antibodies that may be manipulated in position
within a confined fluid and the electric field for controlling
positioning of those charged molecules in lieu of magnetic beads
and magnets. The foregoing process for performing the ELISA (and
ELISA-like) sample analysis and a more detailed description of the
apparatus used to carry out that analysis is presented in the
embodiments illustrated in FIGS. 3-5, the description of which
follows in this specification.
[0056] Reference is next made to FIG. 3, which illustrates in block
diagram form a first practical embodiment of the automated system,
referred to as a single stage apparatus. The apparatus includes
four containers or, as variously termed, vessels 13, 15, 17 and 19,
which hold, respectively, the electrically charged 1.degree.
antibody in a liquid solution, the wash solution, the substrate
reporter and the 2.degree. antibody-enzyme solutions earlier
described. The apparatus further includes a region in which to
collect the sample that is to be analyzed, such as an inlet region
or a vessel. Each of the foregoing vessels is connected by
appropriate fluid conduits, the plumbing, to the pumps and valves
unit 23 of the apparatus as illustrated.
[0057] Pump and valve unit 23 houses individual pumps, not
separately illustrated in the figure, for each of the respective
vessels. An air vent 25 and air filter 27 are also plumbed into the
pumps and valves unit. The air vent and air filter provides a vent
to allow air to separate from solutions and/or remove solutions
from tubes. A fluid conduit 29 extends from the pumps and valves
unit into electrochemical cell 31, the vessel in which the
examination is made. Waste conduit 30 extends from cell 31 to an
appropriate sump or sewer, not illustrated, to permit disposal of
the waste of the process. The apparatus includes an electronic
controller 33, which is a programmed microprocessor or
microcontroller, a field voltage supply 32, a field electrode 35
and a potentiostat 37, which are under control of the controller as
represented by the dash lines. The potentiostat is electrically
coupled to a current sensor 28, represented in dash lines, located
inside cell 31. That current sensor is preferably formed of
interdigitated electrodes, earlier described. Electronic controller
33 includes a selector 34 through which the operator may select the
particular antigen for which the analysis of the sample is being
undertaken, a start button 38 and a display 36, preferably a liquid
crystal display ("LCD"), by which the assay may be reported to the
operator.
[0058] Field electrode 35 is of metal. The field electrode is
supported and positioned against examination cell 31 behind sensor
13. The field electrode is supplied with voltage when required by
the field voltage supply 32. Normally, field voltage supply 32 does
not apply voltage to field electrode 35. When voltage supply 32
receives the command from and is energized by the electronic
controller, the field voltage supply generates the voltage that is
applied to the field electrode, and that voltage in turn produces
an electric field directed through the examination cell and through
(and about) the electrodes of the sensor inside that cell. When a
command is received from controller 33 to extinguish the electric
field in examination cell 31, the field voltage supply is
deenergized.
[0059] Potentiostat 37 supplies the voltage to the electrode array,
the sensor 28, that monitors the reporter, described in step 4 of
FIG. 1, earlier described disposed on the bottom or side of
electrochemical cell 31. That is, the sensor carries any extra
electrical current that flows in series through the electrode array
and potentiostat as a result of the "redox recycling" reaction that
takes place during the latter stage of analysis when the enzyme
substrate is cleaved to release the reporter (Step 6 earlier
described in FIG. 1). The potentiostat is also coupled to an input
of the controller 33 and communicates the electrical current levels
that flow through the interdigital array to the controller.
[0060] Electronic controller 33 is a programmed microprocessor,
microcontroller, computer, as may be variously termed, or the like.
The electronic controller controls each of the pumps and valves
housed in unit 23 and controls energization of field voltage supply
32. The controller also enables and receives monitored current
readings from potentiostat 37. Controllers of the foregoing type
are quite small in size and may be housed or embedded in the
structure of one of the units, such as in pumps and valves unit 23
so as to be inconspicuous. The foregoing components may all be
packaged into a small size compact unit that may easily be carried
by an individual. For added portability, the controller and pumps
may be battery operated. Otherwise the apparatus may be supplied
with electrical operating power from the facility in which used or
by a motor generator set.
[0061] Electronic controller 33 includes a memory, not separately
illustrated, such as ROM or EPROM to permanently store the
operating system and the programs as well as temporary memory such
as RAM, not separately illustrated. The principal programs of the
controller are evident from the description of operation that
follows. It will be realized that the controller serves as a
sequencing device for controlling the pumps, as a collection point
for data, and as a calculating machine for analyzing the data and
displaying the result.
[0062] The electrochemical reaction sensor employed in the
apparatus of FIG. 3 may be any type of sensor that supplies
information on the reporter and supplies that information to the
electronic controller. One such sensor applies a given voltage
across at least two spaced electrodes disposed in the
electrochemical cell and senses the level of electric current that
flows between those electrodes. However the preferred sensor is of
the interdigitated array type one that is described in the cited
'16714 PCT application, IPN '870 application and publications cited
in the background to this invention. The interdigitated array
structure is promulgated as being the most sensitive and, hence,
allows better resolution of the data than other known sensors to
date in this application.
[0063] For operation, electrical power is connected to electronic
controller 33. The operator determines the particular bioagent that
is being sought in the sample material, preferably prepares the
sample in accordance with the ultrasonic energy exposure earlier
described, places the sample in a solution in sample collector 21,
and selects the particular bioagent on selector 34. Vessels 13, 15,
17 and 19 are filled with the appropriate ingredients, earlier
described and not here repeated. The operator operates the start
button 38 and in response electronic controller 33 commences the
automatic operation specified in the stored program.
[0064] The program of the controller motivates dispensing the
contents of sample collector 21 into cell 31 by commanding the
controller to briefly energize an electrical pump associated with
the sample collector. The energized pump pumps the sample-in-liquid
through the plumbing, including conduit 29, and into cell 31, which
may be referred to as the examination cell. Concurrently or
subsequently the program motivates dispensing of the contents of
vessel 13, the electrically charged 1.degree. antibodies in liquid,
by commanding the controller to energize a second electric pump
associated with that vessel for a short interval. The second
electric pump pumps the electrically charged 1.degree. antibodies
into examination cell 31. Presuming the suspect bioagent is present
in the sample material previously deposited in the cell, the
bioagent binds to the antibody, as earlier described in STEP 1 of
FIG. 1.
[0065] Although the description of the embodiment refers to
individual pumps to accomplish the prescribed pumping, those
skilled in the art recognize that other less expensive arrangements
may be employed in alternative embodiments that accomplish the
pumping with a configuration of electrically controlled valves and
pumps that allows pumping of fluid from specific vessels as
selected by the controller. For example, a valve could be
associated with each vessel, the controller would select the
particular valve to open, and then cause a pump to operate and draw
the fluid through the valve.
[0066] Returning to the operation, following a short interval the
controller program next commands the washing of the ingredients in
the fluid in cell 31. For the washing operation, the program
commands energization of field voltage supply 32, which supplies
voltage to the electrode 35 to produce an electric field extending
inside of examination cell 31, and commands energization of a third
pump, not illustrated in the figure, referred to herein as the
aspirating pump. The electric field draws the electrically charged
antibodies to one side of the cell, vacating the charged antibodies
from the solution on the other side of the cell. The aspirating
pump connects to a conduit that extends into the vacated side of
the solution, and the third pump aspirates the fluid and expresses
the waste fluid through waste conduit 30. After a suitable interval
the program halts the third pump and energizes a fourth pump that
connects to a second vessel in wash solution 15 and pumps
sufficient clean fluid to replace the fluid that was removed,
completing the wash. The foregoing wash function corresponds to
STEP 2 of FIG. 1. The solution is then agitated to re-suspend the
charged antibodies in the solution as by aspirating a small amount
of fluid from the vessel and then repumping the aspirated fluid
back into the vessel often referred to as an "up-down" of the
solution.
[0067] The foregoing washing procedure may be repeated the number
of times required by the controller program, and the number written
into the program is one that satisfies the requirements of a
particular operator's experience. For purposes of this description,
the washing step is performed once.
[0068] The program then motivates the delivery of the 2.degree.
antibody-enzyme into examination cell 31 by energizing the pump
associated with vessel 19 for a predetermined interval. In the
examination cell, the antibody-enzymes then bind to another region
of the bioagent, producing the 1.degree. Ab/bioagent/2.degree.
Ab-enz complex. The latter is the same as described in STEP 3 of
FIG. 1.
[0069] The electric field produced by electrode 35 draws the
electrically charged 1.degree. Ab/bioagent/2.degree. Ab-enz complex
to sensor 28. This is the same as represented in STEP 5 of FIG. 1.
The program of controller 33 next motivates the delivery of the
substrate reporter in vessel 17 into the solution in examination
cell 31 by commanding energization of an electric pump, not
illustrated in the figure, associated with vessel 17. The pump is
energized for a predetermined interval and pumps the substrate into
the contents within examination cell 31. Cleavage of the substrate
by the enzyme commences.
[0070] As recalled from the preceding paragraphs, the electric
field that extends through the walls of electrochemical cell 31
draws the 1.degree. Ab/bioagent/2.degree. Ab-enz complex to the
surface of the test electrodes of the electrochemical sensor, not
illustrated in this figure, disposed inside cell 31. At that
location adjacent the electrode surface of the sensor, the bound
enzymes cleave the substrate to produce the reporter molecules.
[0071] Sensor 28 monitors the reaction and reports to the
electronic controller 33. In turn, the controller program analyzes
the data obtained. To monitor electric current through the
examination cell the potentiostat applies a voltage across the
spaced interdigitated electrodes, earlier described, which serve as
sensor 28. That applied voltage produces an electrical current that
passes from one spaced electrode, the anode, through the solution
to the other electrode, the cathode. Absent a reaction in the
solution, the electric current attains a certain default or base
value, depending upon the resistivity of the solution. As the
reaction commences to produce the reporter, the resistivity of the
solution decreases, increasing the current. The effect is referred
to by electrochemists as redox recycling. As the reaction continues
producing greater numbers of reporter molecules, the resistivity
changes further, as does the electric current. The rate of change
of the current is a measure of the concentration of the selected
bioagent. Information of the current, whether the information is in
digital form or analog form, is coupled to electronic controller
33, which analyzes the changing data in real time.
[0072] Essentially concurrently with the pumping of vessel 17, the
controller program commences the checking and assembling of the
data on electrical current flow through the sensor by repetitively
checking the current readings supplied by potentiostat 37 over a
predefined interval of time. For example, one hundred readings may
be taken equally spaced over an interval of ten minutes. The data
obtained is temporarily stored in the memory of the electronic
controller. The program then performs a least-square linear
regression analysis of the data and the analysis generates the
slope of the sensor current (e.g., change of current level vs.
time), a number that represents the rate of change of current.
[0073] The electronic controller also stores in memory (ROM or
EPROM) a library of the standards that have previously been
established in the laboratory to identify bioagents or antigens and
the concentration of the respective antigen in a solution by
measuring the rate of change of current that occurs when using the
known electrochemical ELISA procedure. Each antigen or bioagent
produces a rate of change of current that depends on the
concentration of the bioagent in the sample. For any given
combination of recognition molecule(s) and bioagent or other
antigen, a given concentration produces a unique rate of change of
current. The increase in current as a function of time from the
beginning of the chemical reaction to produce the reporter is
essentially linear, and produces a straight line curve of the type
I=at+b, where "t" represents time, "b" is an initial constant, a
number, and "a" is the slope of the line, also a number. The
foregoing slope information and the correlation of that information
to respective concentration levels has been tabulated and serves as
the standards.
[0074] Thus, for each combination of recognition molecule(s) and
bioagent or other antigen that is to be studied, the library, often
referred to as a "look-up table," contains the correlation between
the slope numbers and the concentration levels correlated to those
slope numbers. After concluding the regression analysis and
obtaining the slope number, the controller program checks to
determine which bioagent or antigen was selected by the operator
and then accesses the stored look-up table for the selected
bioagent or antigen. The computer then compares the slope obtained
in the foregoing regression analysis with corresponding slopes
obtained in measurements of standard concentrations. Once the
computer locates the closest match, the computer then displays the
concentration of the antigen on display 36. Optionally, the
computer may be programmed to also display the calculated slope.
Further, since the volume of the electrochemical cell is known, the
computer may also optionally display the total quantity of antigen
in the test sample.
[0075] The foregoing apparatus is recognized as being automatic in
operation, is very "user-friendly" and does not require highly
skilled personnel to operate. Incorporated within a compact housing
and with optional battery or house supply power the apparatus is
portable and suited for use on location.
[0076] Reference is made to FIG. 4 that shows a block diagram of an
alternative embodiment of the apparatus. This second embodiment is
regarded as a two stage apparatus, whereas the apparatus of FIG. 1
is regarded as a single stage apparatus. For convenience, the
components of the embodiment of FIG. 3 that are essentially the
same in structure as those previously described in FIG. 1 are given
an identical denomination. Those components that are changed
slightly are denominated by the same number used for the
corresponding element and the numbers are primed.
[0077] As inspection of FIG. 4 shows that many of the functional
elements of this embodiment are the same as in the prior
embodiment. The components that have been added include a separate
reaction cell or chamber 39, recirculation valve 40, purge valve
41, an additional field voltage supply 42 and associated field
electrode 43, some additional fluid conduits, some additional
outputs and control lines from the electronic controller, and a
slightly changed program for the electronic controller to
accommodate the additional components and functions.
[0078] In this embodiment the reactions and washes are carried out
in a separate vessel, the reaction chamber 39. A separate field
voltage supply 42 and electric field electrode 43 are employed in
connection with the dielectric walled reaction chamber. The
plumbing and pump arrangement also differs. The electronic
controller is programmed to handle the functions that correspond to
steps 1-6 of FIG. 1 and all of the same operation as in the
embodiment of FIG. 3, excepting the cleavage operation that
generates the reporter. In the foregoing field voltage supply 42
and electric field electrode 43 are used the same as that described
for field voltage supply 32 and field electrode 35 in the
embodiment of FIG. 3.
[0079] At the reporting stage in the present embodiment, the
controller opens valves 40 and 41 permitting the now electrically
charged 1.degree. Ab/bioagent/2.degree. Ab-enz complex in solution
to transfer from the reaction chamber 39 into examination cell 31,
and commands field voltage supply 32 to apply the voltage to field
electrode 35 to direct the electric field into cell 31 and through
the sensor 28. The controller then directs the final chemical,
substrate reporter 17, to be pumped via conduit 29 into the
examination cell 31. As in the prior embodiment, electronic
controller 33 senses the electrical current through the sensor and
potentiostat 37, which is changing, determines the rate of change
of current, e.g., the slope, and from that slope determines the
concentration of the bioagent. The controller then displays the
concentration on display 36. Upon conclusion of the examination the
contents of the cell are expressed through conduit 30 as waste.
[0080] The embodiment of FIG. 4 includes some additional features.
Valve 40 is referred to as a recirculation valve. Should the
program call for recirculating the solution, the controller sets
valve 40 to open a path into a circular conduit. An aspirating
pump, not illustrated, located within unit 23 pumps the solution to
mix the solution.
[0081] Valve 41 is referred to as the purge valve. Instead of
commanding that the solution in chamber 39 be pumped into cell 31,
the controller may instead set valves 40 and 41 to open a passage
into conduit 44 and then initiate an electric pump that pumps the
solution in chamber 39 through the valves and out conduit 44.
Conduit 44 leads into conduit 30 and leads to the waste disposal
system.
[0082] Reference is next made to the schematic illustration of FIG.
5, which illustrates another embodiment of the invention, a
variation of the embodiment of FIG. 4, earlier described. For
convenience, the denomination of the components in this embodiment,
which are the same as those used in the embodiment of FIG. 4, are
identified by the same number, with few exceptions. The embodiment
of FIG. 5 contains electrically operated pumps P1, P2, P3, P4, P5
and P6 and a series of electrically operated valves, V2, Vl, V4, V5
and V6, all of which are controlled by the controller 33. The
convention adopted to describe the condition of a valve when
referring to same as either open or closed may be stated briefly.
When a valve outlet (or inlet) is referred to as being "closed,"
the term means that the outlet is blocked so that fluid cannot flow
there through. When the valve outlet (or inlet) is said to be open,
the term means that the outlet (or inlet) is unobstructed, and
fluid is able to flow there through. Each of the foregoing valves
is a two-way valve and contains an inlet and a pair of outlets, one
of the outlets being normally closed, as illustrated by a gap, and
the other of which is normally open, as represented by an unbroken
line. When the valve is energized, the foregoing state of the
outlets reverses.
[0083] Controller 33, display 36 and start switch 38 are
illustrated in block form. The controller outputs to the respective
valves, pumps and positioners are represented by cable outputs N1,
N2 and N3, in which the cable contains the requisite number of
electrical leads, N, for the respective components associated with
the cable. To avoid undue complication to the schematic, the
electrical leads are not extended to the respective controlled
component in as much as those skilled in the art will understand
the connections. Likewise the input lead from the sensor, not
illustrated, disposed in examination cell 31, is only partially
illustrated.
[0084] Pump P2 is associated with vessel 13 and is for pumping the
electrically charged antibodies in liquid solution contained in the
vessel through the valve V2 and, when the valve is energized, the
plumbing lines into reaction cell 39. Valve V2 is a two-way valve.
The valve contains a normally closed passage that leads into a
conduit that in turn terminates in vessel 13, forming a
recirculating fluid loop in the system. Thus, when pump P2 is
energized by the controller, and valve V2 remains deenergized, such
as illustrated in the figure, the charged antibody solution is
pumped through the recirculating loop. The recirculation of the
charged antibody solution helps to homogenize the distribution of
the antibodies in the solution. When both pump P2 and valve V2 are
energized, the valve opens the recirculating loop and closes the
passage through the conduit into reaction cell 39.
[0085] Pumps P3 and P4 are associated with vessels 15A and 15B. The
two vessels contain different wash solutions, as example, phosphate
buffered saline solution in 15A and Bovine Serum Albumin ("BSA"),
respectively. The BSA is a main component of cow blood in water, a
random protein that prevents the charged antibodies from competing
for binding sites. Thus instead of a single wash in this
embodiment, a double wash with different washing solutions is
accomplished. When commanded by the controller, pumps P3 and P4
will respectively pump the contents of vessels 15A and 15b through
respective conduits into reaction cell 39.
[0086] Pump P5 is associated with vessel 19 containing the
2.degree. antibody-enzyme and pumps the contents into the foregoing
cell via a separate conduit into the reaction cell. Pump P6 is
associated with vessel 21 in which the sample of bioagent is placed
in liquid solution. The pump pumps the sample solution through a
separate conduit into the reaction chamber.
[0087] The enzyme substrate (PAP-GP) is contained in vessel 17.
Valve V6 contains a normally closed inlet, a normally open inlet
and an outlet. The normally open inlet connects via a conduit to
vessel 17 and opens in the bottom side of that vessel. The normally
closed inlet connects via a conduit to an aspiration tube that is
disposed in reaction cell 39. The outlet of the valve connects
through a conduit to the upper end of the examination cell 31. The
examination cell contains an outlet at the bottom end of the cell
that connects via a conduit to a normally open inlet of Valve V4
and to a standpipe A1 that opens to the atmosphere. The foregoing
conduit also includes a flow restrictor R1.
[0088] Each of valves V4 and V5 contain a normally open inlet, a
normally closed inlet and an outlet. Valve V1 contains an inlet, a
normally closed outlet and a normally open outlet. Pump P1 is
connected by conduit in series between the outlet of valve V4 and
the inlet of valve V1. The normally open outlet of valve V4
connects to the outlet of valve V5 and the normally open inlet of
valve V5 connects via a trap and conduit to a second aspiration
tube that extends into the reaction cell 39.
[0089] Assuming that the stage of operation of the foregoing system
is ready to examine for the bioagent in examination cell 31, the
solution located in examination cell 39 must be transferred into
the examination cell 31 and the enzyme substrate (PAP-GP) must be
added thereto. The transfer is accomplished by aspirating the
solution from the examination cell by operating pump P1 and valve
V4. In operating pump P1 creates an aspirating force inside cell 31
through the now closed inlet of valve V4 and the conduit into
expelling gas and/or fluid through the inlet and normally open
outlet of valve V1. The short closed fluid tube A1, referred to as
an accumulator, is also connected in common with the normally
closed passage in valve V4. The accumulator is filled with air and
serves as an "air spring" that evens out the flow rate of the
solution to a uniform slow fluid motion. The draw pulls solution
from reaction cell 39 via the aspirating tube, the normally open
inlet of Valve V6 and the outlet of that valve and into cell 31.
The amount of time required to pump and adequately fill the
examination cell is pre-calibrated during the design of the system
and is known to the program in the controller.
[0090] When the foregoing transfer is completed, the controller
then additionally energizes valve V6. With energization, the
normally closed valve inlet of Valve V6 is switched to open (and
vice-versa for the normally open valve inlet). Pump P1 aspirates a
portion of the contents of cell 31 containing the sample while
drawing the PAP-GP from vessel 17 through valve V6 and into cell
31.
[0091] The foregoing system operates essentially the same as
previously described for the preceding embodiment. Prior to
operating valve V6, the controller readies the examination cell for
detection of the redox recycling that is expected to occur. Thus,
controller 33 commands field voltage supply 32 to energize and
apply the appropriate voltage to the electrode 35 and establish the
appropriate electric field within examination cell 31 with that
field passing through the electrochemical sensor, not illustrated
in the figure, inside the cell. When the PAP-GP is subsequently
introduced into cell 31, the sensor will monitor the current levels
over a period of time, reporting the current levels to electronic
controller 33. As in the prior embodiments, the controller
determines the concentration of the bioagent and displays the
result on display 36.
[0092] Valve V4 is used to determine the flow speed of fluid
through cell 31 by interposing a restrictor R1 and parallel
accumulator A1. High flow rates are desirable for flushing the cell
after a test. Low rates are better when introducing the charged
antibodies so that they are not swept past the electric field by
the force of the flow.
[0093] As one appreciates, the foregoing describes specific aspects
of the mechanization of the ELISA process. The embodiment of FIG. 5
automatically carries out the same functions as earlier described
for FIGS. 1, 2 and 3 in automatically accomplishing the ELISA
process, which need not be repeated.
[0094] The means for holding the sample solution in the prior
embodiments was referred to as a vessel. It should be understood
that the term vessel in that connection is intended to refer to any
region, pipe, conduit or any other suitable means for holding the
sample consistent with the described operation, and is to be not
limited in meaning to a jar or container and may be referred to as
a sample holding means.
[0095] The foregoing embodiments were described using antibodies as
the recognition molecule for suspect bioagents. As is appreciated
it is also desirable on occasion to be able to detect other agents,
such as nucleic acid (e.g., DNA) and proteins, a procedure referred
to herein as ELISA-like since the ELISA procedures are employed for
such detection and the term ELISA may be semantically linked by the
medical researchers to be specific to the use of antibodies. For
those additional agents the recognition molecule used in the
process will likely be a different substance than an antibody. The
foregoing description of the embodiments of the invention, however,
provides the guide to future researchers to find and isolate
appropriate recognition molecules for those additional agents for
use in the practice of the present invention.
[0096] As earlier described the recognition molecule, as example,
may be a monoclonal antibody or, as variously termed, a
single-chain variable fragment ("sFv") molecule. To produce an
amino-acid based charged recognition molecule, here referred to as
a charged antibody, DNA that codes a string of electrically charged
amino acids is spliced onto the coding region of a recognition
molecule, such as a monoclonal antibody or single-chain Fv molecule
("sFv") to produce a chimaeric gene. Amino acids, such as glutamate
or aspartate, will produce a negative electric charge, while basic
amino acids, such as histidine, will produce a positive electric
charge. The particular charge selected depends on the use. The
chimaeric gene can be expressed to generate a fusion protein using
a number of cell-types as expression systems. As one example a
fusion of six glutamates at the carboxyl end of an sFv molecule in
a mammalian cell-culture expression system follows.
[0097] Generation of sFv molecules from a fragment variable ("Fv")
is described in the literature. See Gilliland LK et al., "Rapid and
Reliable Cloning of Antibody Variable Regions and Generation of
Recombinant Single Chain Antibody Fragments, "Tissue Antigens, Vol.
47(1), January 1996, pp. 1-20 and Milenic DE et al., "Construction,
Binding Properties, Metabolism and Tumor Targeting of a
Single-Chain Fv Derived from the Pancarcinoma Monoclonal Antibody
CC49," Cancer Research, Vol. 51(23 Pt 1), December 1991, pp.
6363-71.
[0098] A plasmid is a circular piece of DNA that one is able to
introduce into cells and constitutes a tool to allow genetic
manipulations to be introduced into living cells. The plasmid
contains genes whose expression can be driven to produce a protein
of interest from a living cell. Beginning with a plasmid that
contains an sFv gene of known DNA sequence, two oligonucleotide
primers, short pieces of DNA that possesses complementarity to a
known portion of a larger region of interest in the DNA sequence,
are constructed on the sFv molecule to support a polymerase chain
reaction ("PCR").
[0099] The first primer is generally constructed as the codon ATG
followed by eighteen nucleotides from the amino-terminal sequence
of the sFv molecule. A second primer contains six nucleotides from
the carboxyl-terminal sequence of the sFv followed by a number of
repeats of the codon GAA equal to the number of glutamates to be
added to the molecule, which in this example is six, further
followed by two stop codons (e.g., TGA) and a trailing sequence
(e.g., GAA GAA GAA GAA GAA GAA TGA TGA GGA GAC GGT GAC CAT
GGT).
[0100] A PCR reaction is performed and the resultant product is
cloned in a single step into pCRII-TOPO, a plasmid sold by the
Invitrogen Company of San Diego as a molecular biology research
tool. That cloning procedure and product is described in product
literature, such as published by Invitrogen company of San Diego.
Cloning of monoclonal antibodies is well understood in the art and
for additional details the interested reader may refer to Takahashi
S et al., "Cloning and cDNA Sequence Analysis of Nephritogenic
Monoclonal Antibodies Derived From an MRL/lpr Lupus Mouse,
Molecular Immunology," 1993, Febuary; Vol. 30 (2) pp. 177-182 and
Hong HJ et al, "Cloning and Characterization of cDNAs Coding for
Heavy and Light Chains of a Monoclonal Antibody Specific for Pre-S2
Antigen of Hepatitis B virus."
[0101] The construct, pCRII-TOPO, is then subcloned into an
expression vector, a plasmid that contains a promoter, another
stretch of DNA in which transcription factors bind to drive
expression of a gene of interest cloned into a specific location
and production of the gene's protein product. Subcloning is the
process of cutting a stretch of DNA out of a plasmid using
restriction enzymes that cut DNA only at specific sequences,
referred to as restriction sites, and then cloning that cleaved
stretch of DNA into another plasmid.
[0102] After the gene is subcloned into an expression vector, the
vector is introduced into a cultured cell line (i.e., living cells
held in a bathing medium in a petri dish in an incubator). That
introduction is accomplished by a chemical introduction process,
referred to as transfection, that essentially pokes a hole in the
cells that allow the expression vector, a plasmid, to enter through
the cell membrane and be trapped inside. The holes are small enough
to permit the vector to pass, but not large enough to kill the
cell. Just like a "sick" cell that is infected with a virus is
hijacked by the virus to produce viral proteins, the transfected
cells are hijacked to produce the protein product of the gene
introduced by the expression vector. The protein engineered in the
foregoing way is engineered to be pushed out of the cells into the
bathing medium. In addition to the expressed protein, the bathing
medium for the cells also contains the nutrients that sustain the
living cell, the cell food, and the waste produced by the cell. The
proteins are then extracted from that mixure or "purified."
[0103] One example of a purification process employs a
chromatography column, a hollow tube filled with small bead of a
porous gel. When a solution containing molecules in a variety of
sizes, some larger in size than the pores or holes in the gel and
some smaller, is passed through the gel-filled tube, the small
molecules can pass into the pores in the gel, while the larger
molecules cannot. The smaller molecules will be trapped at least
temporarily in the gel, and will flow more slowly through the
column than the larger molecules. The result is a separation of the
larger molecules based on size. with the larger molecules exiting
the tube first. In that way, the desired protein may be separated
from the liquid and other contents of the bathing medium.
[0104] The preferred purification process is one that takes
advantage of the electrical charge, instead of the foregoing
mechanical one. The charged molecules are smoothly pumped through a
one meter length of tubing at a rate of 10 .mu.l/s. An electrode
extends along the length of the tubing and bears a voltage of 0.7
volts, producing an electrostatic field that extends into the
tubing. The electrode serves as an electrochemical chromatography
column. Charged molecules flowing through the tubing are attracted
by the electrostatic field to the oppositely charged electrode and
are retained, while other contents in the liquid, such as the cell
nutrient and waste product, continue to flow freely through the
tubing. The result is a concentration gradient in the solution with
more charged molecules at the end of the flow stream. This
procedure is repeated on those molecules taken at the end of the
flow stream to further purify or enrich the end product with the
charged molecules; and repeated again until the charged molecules
are sufficiently enriched.
[0105] It is believed that the foregoing description of the
preferred embodiments of the invention is sufficient in detail to
enable one skilled in the art to make and use the invention without
undue experimentation. However, it is expressly understood that the
detail of the elements comprising the embodiment presented for the
foregoing purpose is not intended to limit the scope of the
invention in any way, in as much as equivalents to those elements
and other modifications thereof, all of which come within the scope
of the invention, will become apparent to those skilled in the art
upon reading this specification. Thus, the invention is to be
broadly construed within the full scope of the appended claims.
* * * * *
References